Polycomb group (PcG) proteins are important epigenetic regulators of transcription repression and have long been under active investigation for their roles in Hox gene regulation, X-inactivation, and tumorigenesis in Drosophila and mammalian systems. Recent findings that PcG proteins are conserved in Tetrahymena make it possible to address some of the fundamental mechanisms of Polycomb repression with powerful genetic and biochemical tools available in the unicellular model system. Developmentally regulated heterochromatin formation occurs during the germline-somatic differentiation in Tetrahymena. It has been established that this is an RNAi-dependent PcG-mediated pathway. The proposed studies will explore the mechanism by which the PcG proteins are recruited by RNAi machinery, a mechanism likely to be conserved in higher eukaryotes. We will also examine the potential crosstalk between H3K27 methylation and H2A ubiquitylation, two conserved epigenetic marks deposited by PcG proteins. In particular, we will assess their concerted functions in transcription repression and probe the underlying mechanism. The proposed studies will significantly advance our knowledge for the function of PcG proteins in genome organization and transcription repression. Given that PcG proteins play critical roles in stem cell pluripotency, cell differentiation, and cell malignancy, the proposed studies will also have important implications in public health, including disease diagnosis and treatment. The project matches with the mission of the National Institute of General Medical Science (NIGMS) and is budgeted at $1,250,000 for a five-year period.